A. The SUMO Family of Ubiquitin-Like Proteins
Sentrin-1 (also called SUMO-1) is a protein that can covalently modify specific proteins in a manner analogous to ubiquitination (Okura et al., 1996; Kamitani et al., 1997a; Kamitani et al., 1997b; Matunis et al., 1996; Mahajan et al., 1997; Boddy et al., 1996; Hershko et al., 1998). In mammalian cells, there are three known sentrin family proteins that are expressed in all tissues and appear to have overlapping function (Kamitani et al., 1997a; Kamitani et al., 1997b; Kamitani et al., 1998a; Kamitani et al., 1998b).
In contrast to ubiquitination, sumoylation does not target protein for degradation. Sumoylation, in some cases, actually competes with ubiquitination on the same lysine residues, thus functions almost like an anti-ubiquitin (Desterro et al., 1998). Sumoylation can also alter a protein's cellular localization. For example, sumoylated RanGAP1 is localized in the nuclear envelope, whereas unmodified RanGAP1 is localized in the cytosol (Okura et al., 1996; Matunis et al., 1996). Finally, sumoylation of many transcriptional factors serves to alter their transcriptional activity (Girdwood et al., 2003; Gostissa et al., 1999; Buschmann et al., 2000; Hay et al., 1999; Kirsh et al., 2002; Lehembre et al., 2001; Kishi et al., 2003; Ross et al., 2002; Tojo et al., 2002; Muller et al., 2000).
Sumoylation is a dynamic process that is mediated by activating, conjugating, and ligating enzymes and readily reversed by a family of SUMO-specific proteases (Yeh et al., 2000; Li et al., 2000). In the mammalian system, four SUMO-specific proteases have been reported (Yeh et al., 2000; Gong et al., 2000, Best et al., 2002; Hang et al., 2002; Kim et al., 2000; Nishida et al., 2001; Nishida et al., 2000). SENP1 is a nuclear protease that appears to deconjugate a large number of sumoylated proteins (Gong et al., 2000). SENP2 is a nuclear envelope associated protease that appears to have similar activity as SENP1 when over-expressed (Gong et al., 2000, Hang et al., 2002, Zhang et al., 2002). There is a spliced isoform of mouse SENP2, called SuPr1, which could alter the distribution of nuclear POD-associated proteins, such as CBP and Daxx and converted Sp3 to a strong activator with diffuse nuclear localization (Ross et al., 2002; Best et al., 2002). Two additional SUMO-specific proteases (SENP3/SMT3IP1 and SENP6/SUSP1) have also been reported (Yeh et al., 2000, Kim et al., 2000). SENP3/SMT3IP1 is a nucleolar protein, whereas SENP6/SUSP1 is located in the cytosol (Kim et al., 2000). Although the ability of SENPs to reverse sumoylation is well established, the specificity of each SENP and the difference in the regulatory pathway mediated by each protease remains to be defined.
C. Sumoylation of Androgen Receptor and its Coregulators
Steroid receptors such as the androgen receptor (AR) are ligand-regulated transcription factors belonging to the nuclear receptor superfamily (McKenna et al., 2002). They convey the effects of steroid hormones on the regulation of cell growth, differentiation, and homeostasis (McKenna et al., 2002). To regulate transcription, the receptors bind to specific hormone response elements of target genes and exhibit crosstalk with other transcription factors through protein-protein interactions. A plethora of coregulatory proteins recognized by different functional domains of the receptors, the N-terminal transactivation region, the central DNA-binding domain, and the C-terminal ligand-binding domain, mediate transactivation and transrepression of nuclear receptors. Some nuclear receptors are also known to be ubiquitinated, which targets them for degradation (Lin et la., 2002; Nawaz et la., 1999). Ubc9, the conjugating enzyme for SUMO, has recently been shown to interact with at least two steroid receptors, AR and glucocorticoid receptor (Poukka et al., 1999; Tian et al., 2002). However, coexpressed Ubc9 enhanced AR-dependent transcription in a fashion that appeared to be independent of its ability to catalyze sumoylation (Poukka et al., 1999). AR is sumoylated in vivo at lysine residues 386 and 520 (Poukka et al., 2000). Mutation of these residues increases the transactivation ability of AR, suggesting that sumoylation is involved in the regulation of AR activity (Poukka et al., 2000).
Recently it has been found that four AR coregulators (e.g., p160 family of coactivators, including SRC-1, GRIP1/SRC-2, ACTR/AIB1/RAC3/pCIP, CBP, p300, and pCAF (McKenna et al., 2002)) are sumoylated. SRC-1 has five sumoylation sites and two major sites were localized in NR box situated in the nuclear receptor interacting region 1 (Chauchereau et al., 2003). It was observed that sumoylation can increase interaction of SRC-1 with the progesterone receptor. For the coactivator GRIP1, two residues located in the nuclear receptor interacting region were found to be sumoylated (Kotaja et al., 2002). Substitution of these two sumoylation sites could attenuate the activity of GRIP1 on AR-dependent transcription. HDAC1 and HDAC4 were also found to be sumoylated (David et al., 2002; Colombo et al., 2002; Tussie-Luna et al., 2002). Mutation of two sumoylation sites of HDAC1 profoundly reduced HDAC1-mediated transcriptional repression (David et al., 2002). HDAC4 sumoylation mutant showed a slightly impaired ability to repress transcription as well as reduced histone deacetylase activity (Kirsh et al., 2002).
Despite the lack of a precise understanding of the mechanisms by which androgens act on so many physiological relevant systems, it is readily understood why the AR is an important target in multiple areas of drug discovery and patient therapy. In the oncology area, for example, inhibitors (antagonists or partial antagonists) of androgen receptor function are useful for the treatment of androgen dependent prostate cancer while agonists or partial agonists of the AR are applicable to the treatment of breast cancer and/or prostate cancer. For metabolic and endocrine diseases disorders, agonists or partial agonists of the androgen receptor function are useful for the treatment of age-related diseases and conditions of cachexia in several disease states including, but not limited to, Acquired Immune Disease Syndrome (AIDS). Functional AR has also been identified in various bone cells and, as such, androgen administration has beneficial effects on skeletal development and maintenance in men and women.
E. Pathogenesis of Prostate Cancer
Tumor formation in prostate tissue is accountable for 30% of cancer-related deaths in men. In most case, the disease progresses from a benign hyperplasia to a prostate cancer precursor state referred to as prostatic intraepithelial neoplasia or PIN. PIN formation is replaced with rapidly proliferating prostate cancer cells that readily metastasize to other areas of the body. This atrophy of the prostate gland is attributed to changes at a molecular level. The most reported alteration is the enhanced expression and transcriptional activity of AR. In normal prostate tissue, endogenous androgens bind the AR and prompt translocation of the receptor to the nucleus. The activated AR associates with specific androgen response elements, recruits numerous transcriptional regulators (or AR coregulators), induces transcription of AR-response genes, and thereby promotes prostate growth. Studies in transgenic mice indicate that continuous activation of supra-physiological levels of ARs produces histological signs of prostate cancer as well as visible lesions (Stanbrough et al., 2001). The correlation between enhanced AR-dependent transcriptional activity and prostate carcinogenesis is well accepted; in fact, the expression of the AR-regulated prostate specific antigen (PSA) gene is used as a biological marker for diagnosis of prostate cancer (Culig et al., 2003; DeMarzo et al., 2003). Inhibition of AR activation with androgen ablation is the primary therapy for advanced and metastatic prostate cancer (Scherr et al., 2003). Androgen deprivation initially does induce tumor regression but eventually the tumor becomes unresponsive and averts to an androgen-independent or hormone refractory state.
Similar to androgen-dependent prostate cancer, AR-dependent transcription must be attenuated to arrest tumor proliferation in hormone refractory prostate cancer. However, cellular targets remain to be identified that can regulate AR-dependent transcription irrespective of the mechanism for AR activation (androgen-dependent and/or androgen-independent).